Mitochondrial DNA (mtDNA) molecules from different species of the melanogaster group of the genus Drosophila differ in molecular weight (9.92 x 10 to the 6th power d to 12.35 x 10 to the 6th power d). These differences are almost completely accounted for by differences in the size of a region which denatures at a lower temperature than the rest of the molecule, and therefore appears to be rich in adenine and thymine (A-T). Using electron microscopy, restriction enzyme and cloning technology, ultracentrifugation, thermal melting, base composition, nearest neighbor and pyrimidine tract analyses, we will attempt to elucidate the nucleotide sequence of the largest (3.41 x 10 to the 6th power d) of the A-T rich regions, that of D. melanogaster mtDNA molecules. The occurrence and location throughout the D. melanogaster mtDNA molecules of inverted repeat sequences, ribonucleotides and gene 32 protein binding sites will be investigated. The relative locations of restriction enzyme cleavage sites and the A-T rich region on mtDNAs of D. melanogaster and other selected Drosophila species will be mapped. We will construct heteroduplexes between mtDNA molecules to determine whether the A-T rich regions of the different Drosophila species are homologous. Heteroduplex and thermal melting studies will be carried out to investigate sequence homologies of other regions of mtDNA molecules of the different species. Using the A-T rich region and restriction enzyme cleavage sites as markers, we will continue our electron microscope studies of D. melanogaster mtDNA to gain further information on the mechanisms of replication of this DNA. Efforts will be made to obtain cell cultures of Drosophila species having mtDNA molecules which differ from the mtDNA molecules of D. melanogaster in regard to the size of the A-T rich region and the location of specific restriction enzyme sites. Attempts will then be made to fuse cells from such cultures with D. melanogaster culture cells and to identify recombinant molecules, in order to learn about the mechanism by which exchanges between molecules are made. We will attempt to determine whether low buoyant density, heterogeneously sized circular molecules obtained from lysates of testes represent mtDNA and whether these molecules originate from sperm.